Xanthine oxidase inhibitors

ABSTRACT

Compounds of the following structure are disclosed which are effective inhibitors of the enzyme xanthine oxidase:   IN WHICH Y1 and Y2 are C or N; R1 is H or an alkali metal or ammonium; R2 is H, CH3, a halogen, phenylazo or NO2; R3 is OR1, H, or a halogen; AND R4 is H, NO2 or a halogen. When Y1 is C, Y2 is N and when Y1 is N, Y2 is C.

United States Patent 1191 OBrien et al.

[ Sept. 23, 1975 XANTIIINE OXIDASE INHIBITORS [73] Assignee: ICNPharmaceuticals, Inc., Irvine,

Calif.

22 Filed: Feb. 11, 1974 21 Appl. No.: 440,989

Related US. Application Data [63] Continuation of Ser. No. 172,196, Aug.16, 1971,

abandoned.

[52] US. Cl. 260/256.4 F; 260/154; 424/226; 424/251 [51] Int. Cl. C07D239/00 [58] Field of Search 260/ 256.4 F

[56] References Cited UNITED STATES PATENTS 2,553,500 5/1951 Harsh260/256.4 F 3,244,717 4/1966 Wagner... 260/256.4 F

FOREIGN PATENTS OR APPLICATIONS 375,947 6/1962 Japan 260/256.4 F 76,67710/1970 Germany 260/256.4 F

Primary ExaminerDonald G. Daus Assistant ExaminerJames H. TurnipseedAttorney, Agent, or Firm-William E. Thomson; William C. Steffin; KayI-l. Boswell [57] ABSTRACT Compounds of the following structure aredisclosed which are effective inhibitors of the enzyme xanthine oxidase:

in which Y and Y are C or N;

R, is H or an alkali metal or ammonium; R is H, CH a halogen, phenylazoor N0 R is CR H, or a halogen; and R is H, N0 or a halogen. When Y, isC, Y is N and when Y is N, Y is C.

10 Claims, No Drawings XANTHINE OXIDASE INHIBITORS This is acontinuation, of application Ser. No. 172,196, filed Aug. 16, 1971 nowabandoned.

BACKGROUND OF THE INVENTION It is now well established that the enzymexanthine oxidase is implicated in the production of uric acid by thebody, converting hypoxanthine into xanthine and xanthine, in turn, intouric acid. Under normal conditions, uric acid (2,6,8-trioxypurine) isfound in the body in only small amounts, a concentration in the blood onthe order of about one to about 3 micrograms per 100 milliliters. Undercertain pathological conditions, however, as for example gout, theconcentration of uric acid increases significantly.

Gout, of course, is metabolic disturbance in the body resulting from anoverproduction of uric acid, chronic hyperuricemia (elevated blood uricacid), and progressive accumulation of uric acid in the tissues. Thebody may also progressively lose its capacity to excrete uric acid andis, therefore, in a constant state of uric acid imbalance, accumulatinga greater and greater excess. Its concentration in the blood is high,and, because of its low solubility, it tends to precipitate and formdeposits at various sites where the blood flow is least active,particularly joints and cartilaginous tissues.

One approach to the control of gout commonly used in the past has beenthe prescription of drugs which tended to prevent the accumulation ofuric acid in the body and thus diminish the likelihood of acuterecurrences. Such drugs are identified as uricosuric agents" and promotethe excretion of uric acid in the urine. Examples of such drugs includep-dipropylsulfamyl benzoic acid and sulfinpyrazone. These drugs cannot,however, be administered in conjunction with aspirin or any othersalicylate, which might be given to relieve pain, because the agents andsalicylates are mutually antagonistic, i.e., each tends to ofiset theaction of the other.

A second approach to the treatment of gout which has become popular isthe use of the drug allopurinol,

which blocks the production of uric acid by the body by inhibiting theenzyme xanthine oxidase, which, as noted previously, is responsible forconverting hypoxanthine into xanthine and xanthine into uric acid. Whileallopurinol is effective to inhibit the enzyme xanthine oxidase,nevertheless there are disadvantages which limit its suitability.

First, the toxicity is higher than desirable, having a lethal dosagelevel, LD (the dose required to kill 50% of a group of mice in 2 weekswhen injected into the intraperitone'al cavity) on the order of 150milligrams per kilogram of body weight. Moreover, allopurinol isgradually metabolized in vivo to 4,6-dihydroxy pyrazolo[3,4-d1pyrimidine, which is not as inhibitor as is allopurinol. Inaddition to the foregoing disadvantages, allopurinol, because of itschemical nature, must compete with xanthine to occupy a place on theenzyme xanthine oxidase in order to inhibit the enzyme and thus preventthe formation of uric acid by the body, which likewise limits itsefficiency. It is also known that acute attacks of gouty arthritis occurin the early treatment with allopurinol. It is accordingly necessary togive colchicine during the initial period of therapy to prevent suchacute attacks. There have also been reports of the development of apruritic rash in some patients and of the occasional occurence ofdrowsiness when allopurinol is administered. In view of the foregoing,it is apparent that xanthine oxidase inhibitors which are of acceptabletoxicity and at the same time possess increased inhibition efficiency ascompared to allopurinol are highly desirable.

SUMMARY OF THE INVENTION The present invention thus relates to xanthineoxidase inhibitors comprising imidazo [l,2,a] and pyrazolo [l,5,a]pyrimidine compounds of the following general structure:

Y, and Y are carbon or nitrogen; R is H or an alkali metal or ammonium;R is H, CH a halogen, phenylazo or N0 R is OR H, or a halogen; and R, isH N0 or a halogen. When Y, is carbon, Y is nitrogen, thereby formingthepyrazolo compounds, and when Y, is nitrogen, Y is carbon, thusproviding the imidazo compounds.

, DETAILED DESCRIPTION OF THE INVENTION The xanthine oxidase inhibitorsof this invention are represented by the foregoing structure. As will beseen from the illustrative examples which follow, such compounds,especially compounds in which R is H or phenylazo, demonstrateinhibitory activity significantly greater than that of allopurinol.Moreover, such compounds are stable to enzymatic oxidation and also arenoncompetitive inhibitors of the enzyme xanthine oxidase.

The method of preparing the compounds of the present invention will bedescribed in detail in certain of the illustrative examples. In general,an efficient and comparatively straightforward process is used for thepreparation of such compounds with the precise process varying dependingupon whether an imidazo or pyrazolo compound is produced.

The imidazo pyrimidines are preferably prepared from an imidazolederivative such as amino imidazolinium sulfate, rather than by the morecom- .mon synthetic procedure for the preparation of imidazo pyrimidinesinvolving the condensation of aminopyrimidine derivative with ana-halocarbonyl hyde, only polymeric materials are obtained. This isbelieved to be due to the fact that pyrimidines, with three F wijfi withacidification of the alcoholic mixture with ethanolic hydrogen chlorideto produce the intermediate 2-amino-l -(ethoxymalonyl) imidazole. Whenthis compound is heated'at 135C, it cylcizes to 5,7-dihydroxyimidazo[l,2,a]pyrimidine, which maybe readily purified byrecrystalization from water. Alternatively, the imidazo[ l,2,a]pyrimidine compound may be prepared directly from the imidazolederivative 2-aminoimidazolinium sulfate without isolation of theintermediate compound if the crude sodium salt which separates from theinitial reaction mixture does not come in contact with hot water (inexcess of 70C).

It will, of course, be understood that other of the described5,7-imidazopyrimidines of the present invention can be prepared by theabove-described process if appropriate materials are'u'sed in place ofdiethylmalonate. For the sake of brevity of disclosure, however, thepreparation of such compounds will not be specified in greater detail atthis point; I Treatment of the 5,7-dihydroxy imidazo[ 1,2,a]- pyrimidinewith an aqueous solution of benzene diazonium chloride produces thecorresponding 6-phenylazo derivative. This compound may also be producedby base catalyzed condensation of 2-amino imidazolinium treated withhydrochloric acid, yields 5,7-dihydroxypyrazolo[ l,5,a]pyrimidine. In ananalogous manner, the condensation of 3-amino-4-chloropyrazole, 3-amino-4-bromopyrazole, and 3-amino-4-nitropyrazole with diethylmalonatein the presenceof sodium ethoxide affords 3-chloro, 3-bromo, and3-nit'ro derivatives of 5,7-dihydroxy pyrazolo[l,5,a]pyrimidine.

The condensation of 3-aminopyrazole withimethyl diethylmalonate in thepresence of sodium ethoxide afv fords -5",7 dihydroxy-6-methylpyrazolo[1,5 ,a]pyrimidine. Similarly, the condensation of 2-phenylazodiethylmalonate with 3-amino pyrazole in the presence of sodiumethoxide. yields 5,7-dihydroxy-6- phenylazopyrazolo[ l,5,a]pyrimidine. Y

Refluxing a solution of 5, 7-dihydroxypyrazolo[1.,- 5,a]pyrimidine inphosphorus oxychloride affords 5,7- dichloropyrazolo[1,5,a1pyrimidine(l). The

treatment of (l) .with 2.5 N sodium hydroxide solution yields5-chloro-7-hydroxypyrazolo[ l,5a]pyrimidine (ll). The reductivedehalogenation of (ID-affords 7- hydroxypyrazolo[1,5,a]' pyrimidine(111). This product is identical in allrespects to the product obtainedby the condensation of '3-aminopyrazole with the sodium salt of ethylmalonaldehydic ester in the presence of sodium ethoxide. (reaction 1V).

"The invention will be better understood by reference to the followingspecific but illustrative examples. The ultraviolet spectra was recordedon a Cary-l 5 spectrophotometer. Proton magnetic resonance studies werecarried out with a Hitachi Perkin-Elmer R-20A spectrometer with DSS asinternal reference.

EXAMPLE 1 Preparation of lmidazo [1,2a] pyrimidine 5,7-diol.

, (Method A) I A solution of sodium ethoxide was prepared by dissolvingsodium [8.1g (0.352 formula weights)] in" 200 ml of absolute ethanol.Diethylmalonate [27.43 (0.171 mole)] and 2-amino imidazole hemisulfate[22.2g (0.168 mole)] were added to the sodium ethoxide solution. Theresulting solution was stirred and warmed slowly to reflux. After refluxwas obtained a sodium salt began to separate fromrth'e solution. Themixture was stirred and heated at reflux for 8 hours. After cooling toroom temperature, the sodium salt was separated by filtration, washedwith ethanol, and air dried. The sodium salt was dissolved in ml ofwater (30), and the product precipitated from the solution by theaddition of 6" hydrochloric acid until a pH of 1-2 was obtained; Themixture was cooled to 5 and the product was separated by filtration,washed with ice water, and dried at 100. Recrystallization from waterafforded l1.9g (47 of analytically pure product that had a Mp 360C; Xmax (pH 1) 2l6nm (e 22,200) and 263nm (A, 7,900):- Xmax-"(pl l'l l)223nm" (19,500) and 265nm 7,600). I

" Anal. calcd, for C, 1-1;, N 0,: C, 47.7; H 3.31; N,

27.8... Found: C, 47.8; H, 3.31; N, 27.7.

EXAMPLE 11 Preparation of imidazo [1,2a]-pyrimidine-5,7-diol (Method B)A. 2-Amino-l-(ethoxymalonyl) imidazole A solution of sodium ethoxide wasprepared by dissolving sodium [2.3g co.1 formula weights)] in 150 ml ofabsolute ethanol. Diethylmalonate [8.8g, 55 m moles] and2-aminoimidazole, hemisulfate [6.7g, 50 m moles] were added to thesodium ethoxide solution. The solution was stirred and slowly warmed toreflux, at which time a sodium salt began to precipitate. The mixturewas refluxed for 6 hours and then 50 ml of anhydrous ethanolic hydrogenchloride was added dropwise. After the addition was complete the mixturewas refluxed for 20 minutes, filtered, and the filtrate evaporated todryness. The gummy residue was dissolved in 30 ml of water and the pH ofthis solution brought to 6 by the addition of saturated sodium carbonatesolution to afford a light yellow product. The product was separated byfiltration, washed with water, and dried at 80. Recrystallization ofthis product from water afforded 3.35g (34%) of analytically pureproduct that had a melting point of 175 (slowly melts and resolidifys).

Anal. calcd. for C H N C, 48.7; H, 5.58; N, 21.3. Found: C, 48.7; H,5.69; N, 21.4.

B. imidazo [1,2a] pyrimidine-5,7-diol. 2-amino-1- (ethoxymalonyl)imidazole [1.97g, 10 m moles] was heated at 200 for 45 minutes. Theresidue after cooling was recrystallized from water to afford 1.13g(75%) of analytically pure imidazo [1,2a] pyrimidine-5.7-diol; Mp 360.The ultraviolet absorption spectra of this product is identical to theultraviolet absorption spectra of the product obtained by Method A.

Anal. calcd. for C H,, N 0 C, 47.4; H, 3.31; N, 27.8. Found: C, 47.4; H,3.43; N, 27.5.

EXAMPLE Ill Preparation of 6-Bromoimidazo [1,2a] pyrimidine- 5,7-diol.

Bromine [0.bg (0.00375 formula weights)] in 10 ml of acetic acid wasadded dropwise to a solution of imidazo [1,221] pyrimidine-5,7-diol[0.5g, 3.3 m moles] in 20 ml of acetic acid with good stirring at roomtemperature. After the addition was complete, the mixture was stirred atroom temperature for 3 hours. The solid was separated by filtration,washed with ethanol 2(25 ml), and dried to afford 0.68g (90%) ofproduct. Recrystallization from water afforded in analytically puresample that decomposed above 225C. Amax (pH 1) 220 nm (e, 27,100) and276 nm (e, 9,900) :pmax (pH 11) 226 nm (e,33,600) and 274 nm (e, 7,800)

Anal. calcd. for C H, N, O Br c, 31.3; H, 1.69; N, 18.3. Found: C, 31.2;H, 1.68; N, 18.0.

EXAMPLE lV Preparation of 6-Methylimidazo [1,2a] pyrimidine-5,7-diol.

A solution of sodium ethoxide was prepared by dissolving sodium [1.84g(0.08 formula weights)] in 100 m1 of absolute ethanol. Diethyl methylmalonate [6.96g, 40 m moles] and Z-aminoimidazole, hemisulfate [5.28g,40 m moles] were added to the sodium ethoxide solution. The mixture wasrefluxed with stirring for 8 hours, and then evaporated to dryness atreduced pressure. The residue was dissolved in 100 ml of water (30) andthe ph adjusted to 2 by adding 6 hydrochloric acid. The product wasseparated by filtration, washed with cold water, and recrystallized fromwater to yield 2.74g (40%) of analytically pure product that had amelting point of 310-2 (dec.); umax (pH 1 218 nm (e, 28,800) and 275 nm(e, 1 1,900); umax (pH 11) 223 nm (e, 28,000) and 276 nm (6, 11,700).

Anal. calcd. for C H, N 0 C, 50.9; H, 4.24; N, 25.4. Found: C, 50.9; H,4.38; N, 24.9.

EXAMPLE V Preparation of 6Nitroimidazo 1 ,2a] pyrimidine-5,7-diol.

A solution of sodium ethoxide was prepared by dissolving sodium [4.6g(0.2 formula weights)] in 250 ml of absolute ethanol. Diethylnitromalonate [20.5 g (0.1 mole)] and 2-aminoimidazole, hemisulfate[13.2g (0.1 mole)] were added to the sodium ethoxide solution. Themixture was refluxed with stirring for 9 hours, and then evaporated todryness at reduced pressure. The residue was dissolved in 250 ml ofwater, and the pH adjusted to l-2 by the addition of 6N hydrochloricacid. The product was separated by filtration, washed thoroughly withwater and dried. Purification was afforded by reprecipitating theproduct from dilute sodium hydroxide solution by the addition of 6Nhydrochloric acid to afford 5.9g (30%) of analytically pure product. Mp,3057(dec); A max (pH 1) 207 nm (e, 23,300) and 320 nm (e, 6,300); A max(pH 11) 226 nm (e, 13,300) and 342 nm (5, 4,900).

Anal. calcd. for C H N 0; C 36.7; H, 2.04; N, 28.6. Found: C, 36.8; H,2.16; N, 28.4.

EXAMPLE Vl Preparations of 6-phenylazoimidazo [1,2a] pyrimidine5,7-diol.

A. A solution of sodium ethoxide was prepared by dissolving sodium[1.98g (0.086 formula weights)] in ml of absolute ethanol.Diethylphenylazomalonate [l 1.35g, 43 m moles] and 2-aminoimidazole,hemisulfate [5.68g. 43 m moles] were added to the sodium ethoxidesolution. The mixture was refluxed with stirring for 8 hours, and thenevaporated to dryness at reduced pressure. The residue was dissolved in300 ml of water and the pH adjusted to 5-6 by the addition of a glacialacetic acid. The product was separated by filtra' tion, washed withwater, and purified by recrystallizing from ethanol to afford 3.40g(31%) of analytically pure product that had a melting point of 2779(dec): A max (pH 1) 205 nm (6, 23,000) and 408 nm (e, 27,800); A max (pH11) 217 nm (5, 19,400) and 384 nm (e, 20,900).

Anal. calcd. for C H N 0 C, 56,5; H, 3,53; N, 27.4. Found: C, 56.7; H,3.62; N, 26.6.

B. A solution of benzenediazonium chloride was prepared by treating asolution of aniline [0.3lg, 3,3 m moles] in 7 ml of 1.7N hydrochloricacid with a solution of sodium nitrite [0.25g, 0.0036 formula weights]in 5 ml of water at 5. The benzene diazonium chloride solution was addeddropwise to a stirred solution of imidazo [1.2a] pyrimidine-5,7-diol[0.5g, 3.3 m moles] in 10 ml of 1.25 N sodium hydroxide solution at 10.After the addition was complete, the mixture was stirred at 10 for 30minutes and then at room temperature for 30 minutes. The solid wasseparated by filtration, and purified by reprecipitating from a dilutesodium hydroxide solution by the addition of acetic acid.

EXAMPLE VII Preparation of Pyrazolo [1,5a] pyrimidine-5,7-diol.

This compound was prepared by the procedure of Y. Makisumi as describedin the Chemical and Pharmaceutical Bulletin (Tokyo), 10, 612 (1962).This procedure is as follows:

A solution of sodium. ethoxide was prepared by dissolving sodium [4.6g(0.2 formula weights)] in 300 ml of absolute ethanol. Diethylmalonate[16.0g, 0.1 mole] and 3-aminopyrazole [8.3g, 0.1 mole] were added to thesodium ethoxide solution. The solution was stirred and slowly warmed toreflux at which time a sodium salt began to precipitate. The mixture wasrefluxed for 6 hours and then allowed to cool to room temperature. Thesodium salt was separated by filtration, washed with absolute ethanol,and dried. The sale was dissolved in 250 ml of water and the productprecipitated by the addition of 6 N hydrochloric acid until a pH of l2was obtained. The product was separated by filtration, washed with thewater, and recrystallized from water to afford 7.4g (49%) ofanalytically pure product; mp 2478 (dec); A max (pH 1) 218 nm (6,14,700) and 276 nm (6, 7,300); 229 nm (e, 22,200) and 270 nm (e,10,900).

Anal. calcd. for C H; N C, 47.7; H, 3.31; N, 27.8. Found: C, 47.6; H,3.42; N, 27.7.

EXAMPLE VllI Preparation of 6-Methylpyrazo1o [1,5a] pyrimidine-5,7-diol.

A solution of sodium ethoxide was prepared by dissolving sodium [2.3g(0.1 formula weights)] in 150 ml of absolute ethanol. Diethylmethylmalonate [8.7g, 50 m moles] and 3-aminopyrazole [4.15g, 50 mmoles] were added to the sodium ethoxide solution. The mixture wasrefluxed and stirred for 3 hours, and then evaporated to dryness atreduced pressure. The residue was dissolved in 125 ml of water and thepH adjusted to 1-2 by the addition of 6 N hydrochloric acid. The productwas separated by filtration, washed with water, and dried at 100.Purification was afforded by reprecipitating the product from dilutesodium hydroxide solution by the addition of 6 N hydrochloric acid toafford 4.37g (54%) on analytically pure product; mp, 35860 dec; A max(pH 1) 209 nm (6, 21,300) and 275 nm (e, 10,700); A max (pH 11 226 nm(6, 22,700) and 280 nm (e, 13400).

Anal. calcd. for C, H, N 0 C, 50.9; H, 4.24; N, 25.4. Found: C, 50.9; H,4.36; N, 25.1.

EXAMPLE IX Preparation of 6-Phenylazopyrazolo [1,5a]

pyrimidine-5,7 -diol.

A solution of sodium ethoxide was prepared by dissolving sodium [l,52g(0.066 formula weights)] in 150 ml of absolute ethanol. Diethylphenylazomalonate [8.7lg, 33 m moles] and 3-aminopyrazole [2.74g, 33

m moles] were added to the sodium ethoxide solution. The mixture wasstirred and heated at reflux for 6 hours, and then evaporated to drynessat reduced pressure. The residue was'di ssolved in 300 ml of water andthe pH adjusted to 2 :the addition of 6 N hydrochloric acid. The solidwas separated by filutration, washed with water and ethanol, and airdried. Purification was afforded by reprecipitating the product from adilute sodium hydroxide solution by the addition of 6 N hydrochloricacid to afford 2.34g (28%) of analytically pure product; mp. 300-2(dec); A max (pH 1) 202 nm (5, 25,200), 246 nm (6, 9,700), and 406 nm(e, 30,100 A max (pH 11) 210 nm (6, 19,900) and 386 nm (6, 20,000).

Anal. calcd. for C H N 0 C, 56.5; H, 3.53; N, 27.5. Found: C, 57.0; H,3.84; N, 27.9.

EXAMPLE X Preparation of 3-Chloropyrazolo [1,5a] pyrimidine-5 ,7-diol.

A solution of sodium ethoxide was prepared by dissolving sodium [0.98g(0.0428 formula weights)] in 50 ml of absolute ethanol. Diethylmalonate[3.50g, 21.9 m moles] and 3-amino-4-chloropyrazolo [2.30g, 2.14 m moles]were added to the sodium ethoxide solution. The mixture was stirred andslowly warmed to reflux. After refluxing for 8 hours the mixture wasallowed to cool to room temperature and the sodium salt separated byflltration. The sodium salt was dissolved in 100 ml of water and the pHadjusted to l-2 bythe addition of 6 N hydrochloric acid. The product wasseparated by filtration, washed with ice water, and dried.Recrystallization from water afforded 1.48g (37%) of analytically pureproduct that had a melting point of 270-2 (dec);

A max (pH 1) 219 nm (e, 32,140) and 286 nm (6, 16,680); A max (pH 11)231 nm (e, 47,000) and 275 nm (6, 18,520).

Anal. calcd. for C H; Cl N 0 C, 38.8; H, 2.16; N, 22.6. Found: C, 38.6;H, 2.46; N, 22.9.

. EXAMPLE Xl Preparation of 3-Nitropyrazolo [1,5a] pyrimidine-5,7-diol,hernihydrate.

- The mixture was stirred and refluxed for 16 hours and then allowed tocool to room temperature. The sodium salt was separated by filtration,washed with absolute ethanol and air dried. The sodium salt wasdissolved in m1 of water and the pH of this solution adjusted to 1-2 bythe addition of 6 N hydrochloric acid. The product was separated byfiltration, washed with cold water, and recrystallized with water toafford 3.0g (77%) of analytically pure product; Mp, 2268; A max (pH 1)218 nm (e, 17,600) and 319 nm (6, 8,700); Amax (pH 11) 227 nm (e,12,700) and 332 nm (e, 13,700).

Anal. calcd. for C H, N; Oil 1/2H O: C, 35.1; H, 2.45; N, 27.3. Found:C', 35.1; H, 2.55; N, 27.6.

EXAMPLE xn' A solution oflsodium ethoxide was prepared by dissolving (0.1 20 formulaweights)] in 50 m1 .ofabsolute ethanol. Diethylmalonate[9.6g, 60.2 m

moles] and 3-amino-4-bromopyrazole [9.75g (60.2 m moles)] were added tothe sodium ethoxide solution. The mixture was stirred at roomtemperature for 3 days, and the sodium salt separated by filtration. Thesodium salt was dissolved in 100 ml of water and the pH of this solutionadjusted to 1-2 by the addition of 6N hydrochloric acid. The product wasseparated by filtration, washed with cold water, and dried. Purificationwas accomplished by reprecipitating the product from a dilute sodiumhydroxide solution by the addition of 6 N hydrochloric acid. Theanalytically pure material weighed 4.3g (31%) and decomposed withoutmelting at 265-7; A max (pH 1) 221 nm (6, 20,300) and 283 nm (e, 9,870);A max (pH 1) 233 nm (a, 32,800) and 273 nm (e, 12,850).

Anal. calcd. for C H Br N C, 31.3; H, 1.72; N, 18.2. Found: C, 31.2; H,1.84; N, 18.3.

EXAMPLE XIII Preparation of S-Chloropyrazolo [1,5a] pyrimidine-7-ol A.5,7-Dichloropyrazolo [1,5a] pyrimidine A mixture of pyrazolo[1,5a]-pyrimidine-5,7-diol (32g) and phosphorus oxychloride 160 ml) wasstirred and heated at 1 10. At the end of 1 hour a complete solution wasobtained, and this solution was heated for an additional 1% hours. Theexcess phosphorous oxychloride was removed at reduced pressure using thesteam bath as the source of heat. The red syrup was added slowly to 400gof crushed ice with good stirring. This mixture was stirred at for 30minutes, and then extracted with anhydrous ether 6(400ml). The etheralextract was washed with water, washed with dilute so dium bicarbonatesolution, and dried over anhydrous sodium sulfate. Evaporation of theetheral extract afforded 23g of slightly yellow product, which wassublimed at reduced pressure (1 mm) and elevated temperature (70-100) toafford 11.9g (29%) of analytically pure product that had a melting pointof 72-4; )t max (CH OH) 235 nm (e, 32,000) and 290 mm (6, 2,200).

Anal. calcd. for C H C1 N zC, 38.3; H, 1.59; N, 22.3. Found: C, 38.2; H,1.78; N, 22.4.

B. 5-Chlor0pyrazo1o [1,5a] pyrimidine-7-o1 A A solution of5,7-dichloropyrazolo [1,5a] pyrimidine [6.0g 31.9 m moles] in 1.25Nsodium hydroxide solution 120 ml) was heated at 95 for 1 hour. The hotsolution was treated with decolorizing carbon and filtered. Acificationof the filtrate with 6 N hydrochloric acid until a pH of 2 wasobtainedfafforded the desired product. Recrystallization from a mixtureof water and ethylalcohol (1:1) gave 483g (89%) of analytically pureproduct that had a melting point of 2958 (dec); A max (pH 1) 223 nm (e,34,000) 263 nm (a, 4,900) and 305 nm (6, 5,100); Xmax (pH 11) 227 nm (6,18,200), 281 nm (a, 8,000) and 303 nm (e, 7,300).

Anal. calcd. for C H, N 0 Cl: C, 42.5; H, 2.35; N, 24.8. Found: C, 42.3;H, 2.76; N, 24.7.

XIV

Preparations of Pyrazolo [1,5a]-pyrimidine-7-o1.

A. A mixture of 5-chloropyrazolo [l,5a]-2- pyrimidine-7-ol [8.50g 50 mmoles]; 150 ml of methanol, 10 ml of 1.25 N sodium hydroxide solution,and 10% palladium or charcoal catalyst was placed in a Parohydrogenation apparatus and hydrogenated at a pressure of 42'lbs/inWithin 5 hours, the calculated quantity of hydrogen (Ca. 4.5 lbs./in hadbeen absorbed. The mixture was filtered and the filtrate acidifiedwith'6 N hydrochloric acid until a pH of 2 was obtained. The acidifiedsolution was evaporated to dryness and the residue washed with coldwater. The white crystalline product was recrystallized from a mixtureof methanol and water (2.5:1) to afford 3,13g (46%) of analytically pureproduct that had a melting point of 327-9 (dec); )t max (pH 1) 216 nm(6, 10,600), 260 nm (:2, 6,900) and 295 nm (e, 4,900); A max (pH 11) 226nm (e, 4,900), 280 nm (5, 8,600) and 308 nm (6, 7,700).

Anal. calcd. for C H N 0: C, 53.3; H, 3.70; N, 31.1. Found: C, 53.3; H,3.97; N, 30.9.

B. A mixture of ethylacetate [17.8g, 0.2 mole] and ethylformate [14.8g(0.2 mole] was added dropwise to a suspension of finely cut sodium [4.6g(0.2 formula weights)] in 500 ml of absolute ether with vigorousstirring. After the addition was complete the mixture was stirredvigorously for 20 hours, during which time the sodium salt ofmalonaldehydic ester precipitated. A solution of 3-aminopyrazole [16.6g,0.2 mole] in 250 m1 of absolute ethanol was added to the suspension, andthe resulting mixture heated at 70 until the ether had evaporated. Theresulting mixture was refluxed for 8 hoursand then evaporated to drynessat reduced pressure. The residue was dissolved in 200 ml of water andthis solution treated with decolorizing carbon and filtered. Thefiltrate was acidified to pH2 by the addition of 6 N hydrochloric acid,and the precipitated product separated by filtration. Recrystallizationfrom a methanol and water mixture afforded 3.1g (1 1%) of analyticallypure product that had a melting point of 3258 (dec). The ultra-violetabsorption spectra of this compound is identical to the spectra of thecompound prepared by Method A.

EXAMPLE XV introduced into the incubation mixture containing 150micromoles of tris HCl buffer (pH 7.83), 0.6micromoles EDTA, 0.013micromoles xanthine, and 20 to 40 micrograms of xanthine oxidase in afinal volume of 3.0 milliliters. The xanthine oxidase was purified milkxanthine oxidase from Worthington Biochemical Corporation and had aspecific activity of 0.29 u/mg of protein. The assay was carried out bymixing all the components in a cuvette at 25C. The reaction was startedby the addition of the enzyme with the aid of a stirrer-adder. Changesin OD. were measured for several minutes, and the rate (A OD. perminute) was calculated from the spectral change over the first 15seconds.

ln order to fully evaluate the inhibitory activity and to compare therelative inhibitory abilities of the compounds of this invention withallopurinol, experiments were carried out in which the concentration ofinhibitor was varied over a range of approximately 10' to about l molarand the initial velocity of xanthine oxidation was measured. From a plotof the log of inhibitor concentration against the percent inhibition,the concentration of inhibitor giving 50% inhibition, (1,), wascalculated. This value was obtained from a linear regression analysis ofthe straight lines obtained in this graph. The data is presented inTables I and II which follows as (I/S) values. The lower the (1/8)value, the more potent the inhibitory capability of the compound.

TAB LE I lmidazo [l,2.a]pyrimidines Compound No. R, R, R, R, (US

l H H OH H 3 .57 (2) NH, H OH H 100. l

(3 H CH OH H l .54 (4) H Br OH H 0.92 (5) H phenyl- OH H 0.17

azo (6) NH, phenyl- OH H 55 azo (7 NH, Br OH H 7.65 (8) H N0, OH H 2.80(9) Allopurinol 0.58

' Included for comparison only; outside scope of invention.

TABLE ll Pyrazolo l.5.a]pyrimidines Compound I No. R, R, R, R, (l/S l HH OH H 0.447 (2) H CH, OH H 5 .37 (3) H phenyl- OH H 3 .22

azo (4) H H H H l .08 (5) H H Cl H 2.42 (6) NH, H Cl H 32 7) NH, H OH H33 .4 8) Allopurinol 0.58

Included for comparison only; outside scope of invention.

Referring now more particularly to Table I, it will be observed thatCompounds (l), (3), (4), (5), and (8) demonstrated significantinhibitory activity. Compound (5), the 5,7-dihydroxyimidazo compound inwhich R is phenylazo, demonstrated exceptional activity, beingapproximately 3% times superior to allopurinol, which is also includedin Table I for comparison. It will also be observed that Table 1includes compounds in which an amino group is present at the 7 position.A comparison of the activity results between Compounds (1) and (2), (5)and (6), and (4) and (7), shows that the inhibitory activity isdecreased significantly. A comparison of Compounds l) and (7) and (5)and (6) in Table II shows similar results.

It should also be noted, with respect to Table II, that Compounds (1),(2), (3), (4), and (5) demonstrated significant ability to inhibit theenzyme xanthine oxidase. Compound (I) was especially good, beingsuperior to allopurinol, which has an (l/S) of 0.58.

EXAMPLE XVI Oxidation tests were also conducted on the compounds of thepresent invention to determine their stability against enzyme oxidation.The compounds were tested for their ability to serve as alternatesubstrate in two ways: (1) by substituting the test compound forxanthine in the basic incubation mixture described with respect toExample XV above and measuring the change in 0.D. from 220 to 380 nmover a -minute period, using the repetitive scan accessory of the Caryspectrophotometer, and (2) using the method of Johns, Spector, andRobins, Bioch. P. Col., 18, p. 2371-83 (1969) in which phenozinemethosulfate and cytochrome C were substituted for oxygen as theelectron carrier. Changes in 0.D. of the test compound (Method 1) or ofthe phenozine methosulfate (Method 2) were observed for 30 minutes atthe appropriate wavelength values (550 nm for phenozine methosulfate).

As noted previously, it is known that allopurinol is oxidized to4,6-dihydroxypyrazolo[ 3,4,dlpyrimidine by xanthine oxidase. Because, ofthis, the ability of the compounds of this invention to be oxidized byxanthine oxidase were determined. The reactions were carried out invitro using the methods described above. Under the same conditions,allopurinol was slowly oxidized to 4,6-dihydroxypyrazolo[3,4,d]pyrimidine when oxygen was used to accept electrons and wascompletely oxidized to such compound in the presence of phenozinemethosulfate in less than 5 minutes incubation at 25C.

In contrast, the compounds of this invention shown in Tables I and IIabove were completely stable to enzymatic oxidation under eithercondition.

EXAMPLE XVII The compounds of this invention shown in Tables I and IIwere next subjected to kinetic analysis using the methods of Lineweaverand Burke, or Hanes Enzymes, M. Dixon and E. C. Webb, 1964, Acad. Press,New York, N.Y., to determine the type of inhibition they wereexhibiting. Allopurional has been shown to be a competitive inhibitor ofxanthine oxidase. As can be seen from Tables [I] and IV which follow,the 5,7- dihydroxy compounds having the indicated R, substituents werenon-competitive inhibitors. The K, values shown in Tables Ill and [Vwere calculated from a Dixon-plot (1) against l/v at the two indicatedsubstrate concentrations.

TABLE lll lmidazol l,2,a]pyrimidines Non-inhibited Reaction TABLE IVPyrumlol 1.5.11 [pyrimidine Non-inhihited Reaction Type of R, R- R:, R,Inhibition Km X 10" Vm'" 10 K, 10"

H H OH H Non-Comp. 1.99 l 1.3 6.08, 6.95 H phenyl- OH H 1.10 10.8 101.116

am H CH OH H 3.23 13.3 75.9,9031

EXAMPLE XVIII Using the general procedure and conditions described withrespect to Example XV, the inhibitory activity of the compounds setforth in Table V was determined. The incubation mixture again had atotal reaction volume of 3.0 milliliters and the xanthine concentrationswas 1.32 X 10 M. It will be seen from Table V that the compounds inwhich R, is N C1 or Br, demonstrated highly significant inhibitoryactivity. Also shown in Table V for comparison is Compound (I) of TableII. It will be appreciated that the three other compounds shown in TableV exhibited very good inhibition capability with the halogenatedcompounds showing significantly better Compound (ll of Table n.

(US values than Compounds( 1 It should be further noted that thetoxicity level of the compounds of the present invention is less thanallopurinol. As noted previously, the LD for allopurinol is 150;- 80milligrams per kilogram.

At this point, it should be noted that the chemical structure ofCompound (1) of Table II has been previously reported, Chemical &Pharmaceutical Bulletin (Tokyo), Vol. 10, No. 7, p. 612 et seq. (1962)and Japanese Pat. No. 7983, dated July 1 l, 1962. To our knowledge,however, there is no indication reported that this compound has utilityas an inhibitor of the enzyme xanthine oxidase.

The xanthine oxidase inhibitors are administered as oral preparation, incapsule or tablet form. The tables or capsules will contain from about 5to about 100 milligrams of the inhibitor per tablet or capsule. Therequired dose of the inhibitor, of course, will vary depending upon thecondition of the patient, but will normally range from approximately to600 milligrams per day. To effectively inhibit the enzyme xanthineoxidase, a concentration in the blood from about 1 to about 500micrograms of the inhibitor is required, preferably about 1 to 50micrograms per milliliter.

The capsules will be the usual gelatin capsules and will contain inaddition to the inhibitor, a small quantity, for example less than 5% byweight preferably less than about 1%, magnesium stearate or otherflowing agent, such as Avicel (carboxymethylcellulose). Tablets willinclude the inhibitor and, e.g., a binder, which may be a gelatinsolution, a starch paste in water, polyvinyl pyrrilidone, polyvinylalcohol in water, etc., with a typical sugar coating.

We claim:

1. A compound of the structure I J. p; Y!

wherein Y, and Y, are C or N;

R, is H or an alkali metal or ammonium;

R is H, CH halogen, or N0 R,, is OR,, H, or halogen;

and R,, is H, NO, or halogen; provided that when one of Y, or Y, is C,the other is N;

that when Y, is C, Y, is N and R is H, R,, is H, halogen or OR, where R,is an alkali metal or ammonium and R is H, CH NO, or halogen; and thatwhen Y, is N and Y, is C, R, is H.

2. The compound of claim 1 in which Y, is C and Y, is N, and R,, R, andR are H and R is OH.

3. The compound of claim 1 in which Y, is C and Y, is N, and R,, R andR, are H and R is Cl.

4. The compound of claim 1 in which Y, is C and Y, is N, and R, and Rare H, R, is OH and X is NO,.

5. The compound of claim 1 in which Y, is C and Y, is N, and R, and Rare H, R, is OH and R, is Cl.

6. The compound of claim 1 in which Y, is C and Y, is N, and R, and Rare H, R,, is OH and X is Br.

7. The compound of claim 1 in which Y, is N and Y, is C, and R,, R and Rare H, and R,, is OH.

8. The compound of claim 1 in which Y, is N and Y, is C, and R, and Rare H, and R is CH, and R is OH.

9. The compound of claiml in which Y, is N and Y is C, and R, and R, areH, R, is Br and R,, is OH.

10. The compound of claim 1 in which Y, a N is C, and R, and R are H, Ris N0 and R is OH.

1. A COMPOUND OF THE STRUCTURE
 2. The compound of claim 1 in which Y1 isC and Y2 is N, and R1, R2 and R4 are H and R3 is OH.
 3. The compound ofclaim 1 in which Y1 is C and Y2 is N, and R1, R2 and R4 are H and R3 isCl.
 4. The compound of claim 1 in which Y1 is C and Y2 is N, and R1 andR2 are H, R3 is OH and X is NO2.
 5. The compound of claim 1 in which Y1is C and Y2 is N, and R1 and R2 are H, R3 is OH and R4 is Cl.
 6. Thecompound of claim 1 in which Y1 is C and Y2 is N, and R1 and R2 are H,R3 Is OH and X is Br.
 7. The compound of claim 1 in which Y1 is N and Y2is C, and R1, R2, and R4 are H, and R3 is OH.
 8. The compound of claim 1in which Y1 is N and Y2 is C, and R1 and R4 are H, and R2 is CH3 and R3is OH.
 9. The compound of claim 1 in which Y1 is N and Y2 is C, and R1and R4 are H, R2 is Br and R3 is OH.
 10. The compound of claim 1 inwhich Y1 is N and Y2 is C, and R1 and R4 are H, R2 is NO2 and R3 is OH.